Abstract

Congenital heart defects are responsible for more neonatal deaths than any other birth defect. Although genetic and environmental factors play an important role, either separately or in combination (multifactorial), still the cause in most cases remains unknown.

Changing life styles, e.g. exposure of the mother to excessive alcohol, nicotine in tobacco smoke, easily available traditional and, over the counter medicines and environmental contaminants could be possible causes of congenital malformations. Maternal diseases like diabetes mellitus are also one of the etiological factors for developmental defects. Several developmental genes, for instance connexin 43; one of the key proteins involved in cardiovascular development, and endothelin 1; another important gene required in many developmental processes, could be responsible for developmental anomalies of the heart. Supplementation with micronutrients such as folic acid and Vitamin C during the periconceptional period has been shown to prevent some neural tube and congenital heart defects. This study was aimed at evaluating the adverse effects of ethanol, retinoic acid, nicotine, cadmium chloride, sodium fluoride, ginseng and diabetic conditions on chick cardiomyocytes cultured in the micromass system, and examining the potential protective effects of folic acid and vitamin C. Also teratogenic effects of some of the teratogens, ethanol, nicotine, retinoic acid and diabetic conditions, were examined using in ovo culture.

Hearts were dissected from 5 day old White Leghorn chick embryos and the cells were isolated and cultured. They were exposed to different concentrations of test chemicals. Folic acid and vitamin C were added to see any protective effects. Cell viability was assessed using the resazurin reduction assay and the kenacid blue assay was performed for determining cell number. For in ovo culture, day 3 chick embryos were injected with ethanol, nicotine, retinoic acid or diabetic molecules or a combination of teratogenic chemicals and vitamins (folic acid and vitamin C). Immunohistochemistry and western blotting were employed to detect the expression of connexin 43 and endothelin 1. Results of micromass culture revealed that ethanol, retinoic acid, nicotine, cadmium chloride and diabetic conditions dramatically reduced cellular differentiation, cell viability and protein content in a dose dependant manner. However, vitamin C (100µM) and folic acid (1mM) administered concurrently with these chemicals, except for cadmium chloride, could significantly improve all parameters such that the values were comparable with the control. Nicotine had no effect on cell viability and protein content, but cell beating was significantly affected. This effect was reversed by the addition of Vitamin C and folic acid. Results of in ovo culture showed that ethanol and diabetic conditions caused gross and histological malformations in chick embryos. However their effects were abrogated with supplemental folic acid and vitamin C. Immunohistochemical and western blotting results demonstrated a decreased expression of Cx43 and endothelin 1 in ethanol, retinoic acid, nicotine and diabetic condition treated cells while addition of vitamins restored their expression so they were comparable to controls.

It may be that environmentally induced teratogenic effects on heart development could be prevented by supplementation with Vitamin C and folic acid during pregnancy.